Neurotrophic factors are endogenous proteins that modulate cell signaling pathways regulating stem cell proliferation, neuronal differentiation, differentiation, growth and regeneration (Barde Y, “Trophic factors and neuronal survival”, Neuron 2:1525-1534 (1989); Gotz, R., et al., “The conservation of neurotrophic factors during vertebrate evolution”, Comp Biochem Physiol Pharmacol Toxicol Endocrinol 108: 1-10 (1994); and Goldman, S. A., “Adult neurogenesis: from canaries to the clinic”, J Neurobiol 36: 267-86 (1998)). They are generally small, soluble proteins with molecular weights between 13 and 24 KDa and often function as homodimers. Because of this physiological role, neurotrophic factors are useful in treating the degeneration of nerve cells and the loss of differentiated function that occurs in a variety of neurodegenerative diseases.
Many neurotrophic factors are both neuroprotective (protecting neurons from injury) and neurorestorative (promoting structural and functional regeneration). The best defined protective functions are seen during neural development. During development, excessive numbers of neurons are generated in many brain regions. Developing neurons that fail to make connections with appropriate trophic factor producing target cells are deprived of necessary neurotrophic factors and die. Those neurons that establish connections survive and function properly (e.g. NGF; see Campenot, R. B. and MacInnis, B. L. “Retrograde transport of neurotrophins: fact and function”, J Neurobiol 58: 217-229 (2004)). Neurotrophic factors are also capable of promoting the re-growth of damaged neurons and their processes both in vitro and in animal models (see Lad, S. P. et al., “Individual and combined effects of TrkA and p75NTR nerve growth factor receptors: A role for the high affinity receptor site”, J Biol Chem 278: 24808-24817 (2003a) and Lad, S. P. et al., “Nerve growth factor: structure, function and therapeutic implications for Alzheimer's disease”, Curr Drug Targets CNS Neurol Disord 2: 315-334 (2003b)). Identifying neurotrophic factors with the right combination of protective and restorative actions and developing effective strategies for drug delivery have profound therapeutic implications for Parkinson's disease, Alzheimer's disease, Huntington's disease and other degenerative processes in the brain (including those induced by brain injury).
Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor shown to dramatically protect and enhance the function of dopamine neurons in vitro and in vivo in rodents and monkeys (Beck, K. D., et al., “Mesencephalic dopaminergic neurons protected by GDNF from axotomy-induced degeneration in the adult brain”, Nature, 373:339-41 (1995); and Bjorklund, A., et al., “Towards a neuroprotective gene therapy for Parkinson's disease: use of adenovirus, AAV and lentivirus vectors for gene transfer of GDNF to the nigrostriatal system in the rat Parkinson model”, Brain Res., 886:82-98 (2000), Gash, D. M., et al., “Functional Recovery in Parkinsonian Monkeys Treated with GDNF, Nature”, 380:252-255 (1996); Grondin, R., et al., “Striatal GDNF Infusion Promotes Structural and Functional Recovery in Advanced Parkinsonian Monkeys. Brain”, 125:2191-2201 (2002); Grondin, R., et al., “GDNF Increases Stimulus-evoked Dopamine Release and Motor Speed in Aged Rhesus Monkeys”, J. Neurosci., 23:1974-1980 (2003); Hebert M. A., et al., “Functional Effects of GDNF in Normal Rat Striatum: Presynaptic Studies Using In Vivo Electrochemistry and Microdialysis”, J. Pharm. Exp. Ther., 279:1181-1190 (1996); Hebert M. A. and Gerhardt, G. A., “Behavioral and Neurochemical Effects of Intranigral GDNF Administration on Aged Fischer 344 Rats”, J. Pharm. Exp. Ther., 282: 760-768 (1997); Hou, J. G. G., et al., “Glial Cell line-Derived Neurotrophic Factor Exerts Neurotrophic Effects on Dopaminergic Neurons In Vitro and Promotes Their Survival And Regrowth After Damage by 1-Methyl-4-Phenylpyridinium”, J. Neurochem., 66: 74-82 (1996); Kordower, J. H., et al., “Clinicopathological Findings Following Intraventricular Glial-Derived Neurotrophic Factor Treatment in a Patient with Parkinson's Disease”, Ann Neurol., 46(3):419-424 (1999); Kordower, J. H., et al., “Neurodegeneration Prevented by Lentiviral Vector Delivery of GDNF in Primate Models of Parkinson's Disease”, Science, 290: 767-773 (2000); Palfi, S., et al., “Lentivirally Delivered Glial Cell Line-derived Neurotrophic Factor Increases the Number of Striatal Dopaminergic Neurons in Primate Models of Nigrostriatal Degeneration”, J. Neurosci., 22:4942-4954 (2002); Tomac, A., et al., “Protection and Repair of the Nigrostriatal Dopaminergic System by GDNF In Vivo”, Nature, 373:335-339 (1995)).
The current standard treatment, levodopa, is palliative and does not prevent the relentless progression of Parkinson's degeneration. GDNF exerts effects on dopamine neurons that slow the process of Parkinson's disease and even reverses some of the degenerative changes. Preclinical studies conducted to date suggest that GDNF exerts at least three general trophic actions on dopamine neurons in the substantia nigra: pharmacological upregulation, restoration and neuroprotection. With regard to pharmacological upregulation, GDNF upregulates dopaminergic functions, such as increasing the evoked release of dopamine (Gerhardt, G. A. et al., “GDNF improves dopamine function in the substantia nigra but not the putamen of unilateral MPTP-lesioned rhesus monkeys”, Brain Res 817: 163-171 (1999) and Grondin et al., 2003). It also appears to modulate the phosphorylation of TH (Salvatore, M. et al. “Striatal GDNF administration increases tyrosine hydroxylase phosphorylation in the rat striatum and substantia nigra”. J Neurochem. 90:245-54, (2004)). With regard to restoration, GDNF increases the number of neurons expressing the dopamine markers TH and DAT in the substantia nigra (Gash et al., 1996; Kordower et al., 2000; and Grondin et al., 2002). This suggests that one trophic action is to stimulate recovery of injured/quiescent nigral neurons. Supporting this interpretation is the consistent observation that GDNF promotes increases in dopamine neuron perikaryal size and the number of neurites. With regard to neuroprotection, nigrostriatal administration of GDNF either shortly before or following a neurotoxic challenge (e.g. 6-OHDA, methyl-amphetamine or MPTP) protects dopamine neurons from injury in rodents and nonhuman primates (Kordower et al., 2000 and Fox, C. M., “Neuroprotective effects of GDNF against 6-OHDA in young and aged rats”, Brain Res 896:56-63 (2001)).
Accordingly, GDNF therapy is expected to be helpful in the treatment of nerve damage caused by conditions that compromise the survival and/or proper function of one or more types of nerve cells. Such nerve damage may occur from a wide variety of different causes. Nerve damage may occur to one or more types of nerve cells by, for example: (1) physical injury, which causes the degeneration of the axonal processes and/or nerve cell bodies near the site of injury; (2) temporary or permanent cessation of blood flow to parts of the nervous system, as in stroke; (3) intentional or accidental exposure to neurotoxins, such as chemotherapeutic agents (e.g., cisplatinum) for the treatment of cancer or dideoxycytidine (ddC) for the treatment of AIDS; (4) chronic metabolic diseases, such as diabetes or renal dysfunction; or (5) neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Amyotrophic Lateral Sclerosis (ALS), which result from the degeneration of specific neuronal populations.
GDNF therapy may be particularly helpful in the treatment of neurodegenerative conditions involving the degeneration of the dopaminergic neurons of the substantia nigra, such as Parkinson's disease. The expected impact of GDNF therapy is not just to produce an increase in the dopaminergic neurotransmission at the dopaminergic nerve terminals in the striatum (which will result in a relief of the symptoms), but also to slow down, or even stop, the progression of the degenerative processes and to repair the damaged nigrostriatal pathway and restore its function. GDNF may also be used in treating other forms of damage to or improper function of dopaminergic nerve cells in human subjects. Such damage or malfunction may occur in schizophrenia and other forms of psychosis. The only current treatments for such conditions are symptomatic and require drugs which act upon dopamine receptors or dopamine uptake sites, consistent with the view that the improper functioning of the dopaminergic neurons which innervate these receptor-bearing neuronal populations may be involved in the disease process.
However, initial clinical trials involving ventricular delivery of GDNF showed no statistically significant differentiation of the placebo and active treatment groups (Nutt, J. G. et al. “Randomized, double-blind trial of glial cell line-derived neurotrophic factor (GDNF) in PD”, Neurology 60: 69-73 (2003)), perhaps because insufficient amounts of GDNF reached critical target sites from the CSF (Ai, Y. et al., “Intraputamenal Infusion in Aging Rhesus Monkeys: Distribution and Dopaminergic Effects”, J Comp Neurol 461: 250-26125 (2003); and Kordower, J. H., et al. (2000)). In addition, a phase 2 trial evaluating intraputamenal delivery of glial cell line-derived neurotrophic factor (GDNF) for the treatment of Parkinson's disease failed to achieve its primary end point, a 25% improvement on the Unified Parkinson Disease Rating Scale (UPDRS) motor score “off” medication after six months of treatment (Lang, A. E. et al., “Randomized controlled trial of intraputamenal glial cell line-derived neurotrophic factor infusion in Parkinson's disease”, Ann Neurol 59:459-466 (2006)). There are strong indications from studies in rhesus monkeys using the same delivery system and protocol followed in the phase 2 study that drug bioavailability significantly contributed to the failure of the trial (Salvatore et al., “Point source concentration of GDNF may explain failure of phase II clinical trial”. Exp Neurol 202 (2): 497-505 (2006)). The concentration of GDNF around the catheter tip and limited diffusion into surrounding brain parenchyma was limited to a brain volume representing 2-9% of the human putamen. Thus GDNF distribution in the phase 2 trial was likely limited to a small brain region, and could affect only a limited segment of the brain undergoing parkinsonian degeneration.
Successful trophic factor therapy requires site-specific delivery and distribution of the trophic factor throughout the target tissue (the putamen for Parkinson's disease). The blood brain barrier effectively blocks entry from blood borne proteins, including trophic factors. Infusions into the cerebrospinal fluid are not effective in humans because of brain size and may produce unwanted side effects by stimulating other trophic factor responsive populations such as sensory neurons.
In addition to focal delivery into the appropriate site, the delivery must be tightly regulated. Regardless of the method used to deliver GDNF (i.e., direct infusion, stem cells, encapsulated cells, gene therapy) prolonged elevated levels of GDNF in the brain outside of the target area may produce adverse side-effects. Circulating antibodies to GDNF are one possible outcome and it is quite typical to find antibodies to endogenous proteins used therapeutically (e.g. beta interferon and insulin, see Durelli, L., et al., “Anti-interferon antibodies in multiple sclerosis: molecular basis and their impact on clinical efficacy”, Front Biosci 9: 2192-2204 (2004) and Stoever, J. A. et al., “Inhaled insulin and insulin antibodies: a new twist to an old debate”, Diabetes Technol Ther 4: 157-161 (2002)). The effects of circulating GDNF antibodies are not known. Focal Purkinje cell lesions have been reported in some monkeys receiving high levels of GDNF in a toxicology study (see Sherer, T. B., et al., Movement Dis 21:136-141 (2006)). Another possible side-effect is aberrant sprouting and tyrosine hydroxylase downregulation of the nigrostriatal dopaminergic pathway in rats exposed to high GDNF levels from viral vector gene transfer (Georgievska, B., et al. “Neuroprotection in the rat Parkinson model by intrastriatal GDNF gene transfer using a lentiviral vector”, Neuroreport 13: 75-82 (2002)). Also, increased neuronal death has been reported in rats with elevated GDNF from viral vector gene transfer in a stroke model (Arvidsson, A. et al., “Elevated GDNF levels following viral vector-mediated gene transfer can increase neuronal death after stroke in rats”, Neurobiol Dis 14: 542-556, (2003)).
While GDNF has not met the criteria for clinical efficacy in the two phase 2 trials conducted to date (Nutt et al., 2003; Lang et al., 2006), it appears to be the most potent dopaminergic trophic factor found to date. Thus, the ideal drug for treating Parkinson's disease and other neurodegenerative processes in the brain would possess the positive trophic actions of GDNF. Delivery could be targeted to the appropriate brain area by any of a number of methods including direct infusion, viral vectors or even nasal sprays. In particular, biologically active peptides with trophic actions may offer many of the desired properties. To date, such biologically active peptides have not been identified.
A crude peptide extract from the brain cerebrolysin has been tested in human studies, with modest effects reported (Lukhanina, E. P. et al., “Effect of cerebrolysin on the electroencephalographic indices of brain activity in Parkinson's disease”, Zh Nevrol Psikhiatr Im S S Korsakova 104: 54-60 (2004)). Three small molecule compounds have also been tested in Parkinson's disease patients: the tripeptide glutathione, tocopherol, and Coenzyme Q10 (Weber, C. A., et al. “Antioxidants, supplements and Parkinson's disease”, Ann Pharmacother 40: 935-938 (2006)). The three small molecule compounds also appear to have only minor benefits for patients.
Consequently, there continues to exist a long-felt need for effective agents and methods for the treatment and prevention of brain diseases and injuries that result in dopaminergic deficiencies. Accordingly, it is an object of the present invention to provide agents for treating and preventing such diseases and injuries in a subject, comprising novel amidated GDNF-derived peptides that have dopaminergic trophic factor activity. This and other such objectives will be readily apparent to the skilled artisan from this disclosure.